Near-Infrared In Vivo Imaging of Claudin-1 Expression by Orthotopically Implanted Patient-Derived Colonic Adenoma Organoids.

BACKGROUND: Claudin-1 becomes overexpressed during the transformation of normal colonic mucosa to colorectal cancer (CRC). METHODS: Patient-derived organoids expressed clinically relevant target levels and genetic heterogeneity, and were established from human adenoma and normal colons. Colonoids were implanted orthotopically in the colon of immunocompromised mice. This pre-clinical model of CRC provides an intact microenvironment and representative vasculature. Colonoid growth was monitored using white light endoscopy. A peptide specific for claudin-1 was fluorescently labeled for intravenous administration. NIR fluorescence images were collected using endoscopy and endomicroscopy. RESULTS: NIR fluorescence images collected using wide-field endoscopy showed a significantly greater target-to-background (T/B) ratio for adenoma versus normal (1.89 ± 0.35 and 1.26 ± 0.06) colonoids at 1 h post-injection. These results were confirmed by optical sections collected using endomicroscopy. Optical sections were collected in vivo with sub-cellular resolution in vertical and horizontal planes. Greater claudin-1 expression by individual epithelial cells in adenomatous versus normal crypts was visualized. A human-specific cytokeratin stain ex vivo verified the presence of human tissues implanted adjacent to normal mouse colonic mucosa. CONCLUSIONS: Increased claudin-1 expression was observed from adenoma versus normal colonoids in vivo using imaging with wide field endoscopy and endomicrosopy.

In Vitro Porcine (Explant) Colon Culture.

Models have been extensively used to investigate disease pathogenesis. Animal models are costly and require extensive logistics for animal care, and samples are not always suitable for different analytical techniques or to answer the research question. In vitro cell culture models are generally focused on recreating a specific characteristic of an organ and are limited to a single cell population that does not display the characteristic tissue architecture of the source organ. In addition, such models do not account for the many interactions between pathogens and the diverse cell subsets that are normally present in a given organ. Conclusions based on conventional 2D cell culture methods are limited, requiring extrapolation from a reductionist model to understand in vivo events. In vitro organ culture (IVOC) offers a way to overcome some of these limitations. Explants conserve important in vivo characteristics, such as different cell types and complex tissue architecture. This in vitro (ex vivo) organ culture protocol of the swine large intestine aims at maintaining viable colonic mucosa for up to 5 days. The protocol described herein applies a combination of methods used for immortalized cell culture and stem cell stimulation to support the physiological cellular flow inherent of the intestinal mucosa. Required equipment includes a hyperoxic chamber and culture at the air-liquid interface.

Near-Infrared Imaging of Colonic Adenomas In Vivo Using Orthotopic Human Organoids for Early Cancer Detection.

Colorectal cancer is a leading cause of cancer-related morbidity and mortality worldwide. Premalignant lesions that are flat and subtle in morphology are often missed in conventional colonoscopies. Patient-derived adenoma colonoids with high and low cMet expression and normal colonoids were implanted orthotopically in the colon of immunocompromised mice to serve as a preclinical model system. A peptide specific for cMet was labeled with IRDye800, a near-infrared (NIR) fluorophore. This peptide was administered intravenously, and in vivo imaging was performed using a small animal fluorescence endoscope. Quantified intensities showed a peak target-to-background ratio at ~1 h after intravenous peptide injection, and the signal cleared by ~24 h. The peptide was stable in serum with a half-life of 3.6 h. Co-staining of adenoma and normal colonoids showed a high correlation between peptide and anti-cMet antibody. A human-specific cytokeratin stain verified the presence of human tissues implanted among surrounding normal mouse colonic mucosa. Peptide biodistribution was consistent with rapid renal clearance. No signs of acute toxicity were found on either animal necropsy or serum hematology and chemistries. Human colonoids provide a clinically relevant preclinical model to evaluate the specific uptake of a NIR peptide to detect premalignant colonic lesions in vivo.

A Pilot Study: Transcriptional Profiling, Functional Analysis, and Organoid Modeling of Intestinal Mucosa in Hirschsprung Disease.

BACKGROUND: Hirschsprung disease (HSCR) is a congenital colonic aganglionosis. Many HSCR patients develop enterocolitis despite surgical resection. The pathophysiology of this inflammatory process is poorly understood. We compared transcriptional profiles and function of ganglionic and aganglionic tissue in HSCR patients. METHODS: RNA sequencing was performed on mucosal tissues from HSCR patients (n = 6) and controls (n = 3). Function of matched ganglionic and aganglionic regions were investigated utilizing organoids generated from these tissues. RESULTS: Transcriptional differences observed in ganglionic and aganglionic regions of HSCR patients included upregulation of genes involving inflammation, cell differentiation and proliferation as well as decreased expression of genes encoding mucins compared to controls. Organoids derived from ganglionic and aganglionic regions of HSCR patients were similar in epithelial cell differentiation, epithelial barrier formation and response to stimulation with bacterial metabolites and pro-inflammatory cytokines. CONCLUSIONS: Despite normal ganglionic structure, the section of colon adjacent to the aganglionic region in HSCR patients has perturbed gene expression which resembles the aganglionic segment. Transcriptional and functional changes in colonic epithelium are persevered in the ganglionic colon used for pull-through surgery. This may explain persistence of enterocolitis despite surgical excision of aganglionic colon and subsequent endorectal pull-through performed with ganglionic colon during correction of HSCR. LEVEL OF EVIDENCE: N/A.

Transferrin Receptor-Mediated Iron Uptake Promotes Colon Tumorigenesis.

Transferrin receptor (TFRC) is the major mediator for iron entry into a cell. Under excessive iron conditions, TFRC is expected to be reduced to lower iron uptake and toxicity. However, the mechanism whereby TFRC expression is maintained at high levels in iron-enriched cancer cells and the contribution of TFRC to cancer development are enigmatic. Here the work shows TFRC is induced by adenomatous polyposis coli (APC) gene loss-driven ß-catenin activation in colorectal cancer, whereas TFRC-mediated intratumoral iron accumulation potentiates ß-catenin signaling by directly enhancing the activity of tankyrase. Disruption of TFRC leads to a reduction of colonic iron levels and iron-dependent tankyrase activity, which caused stabilization of axis inhibition protein 2 (AXIN2) and subsequent repression of the ß-catenin/c-Myc/E2F Transcription Factor 1/DNA polymerase delta1 (POLD1) axis. POLD1 knockdown, iron chelation, and TFRC disruption increase DNA replication stress, DNA damage response, apoptosis, and reduce colon tumor growth. Importantly, a combination of iron chelators and DNA damaging agents increases DNA damage response and reduces colon tumor cell growth. TFRC-mediated iron import is at the center of a novel feed-forward loop that facilitates colonic epithelial cell survival. This discovery may provide novel strategies for colorectal cancer therapy.

Mapping the adult human esophagus in vivo and in vitro.

Many esophageal diseases can arise during development or throughout life. Therefore, well-characterized in vitro models and detailed methods are essential for studying human esophageal development, homeostasis and disease. Here, we (1) create an atlas of the cell types observed in the normal adult human esophagus; (2) establish an ancestrally diverse biobank of in vitro esophagus tissue to interrogate homeostasis and injury; and (3) benchmark in vitro models using the adult human esophagus atlas. We created a single-cell RNA sequencing reference atlas using fresh adult esophagus biopsies and a continuously expanding biobank of patient-derived in vitro cultures (n=55 lines). We identify and validate several transcriptionally distinct cell classes in the native human adult esophagus, with four populations belonging to the epithelial layer, including basal, epibasal, early differentiating and terminally differentiated luminal cells. Benchmarking in vitro esophagus cultures to the in vivo reference using single-cell RNA sequencing shows that the basal stem cells are robustly maintained in vitro, and the diversity of epithelial cell types in culture is dependent on cell density. We also demonstrate that cultures can be grown in 2D or as 3D organoids, and these methods can be employed for modeling the complete epithelial layers, thereby enabling in vitro modeling of the human adult esophagus.

Rapid establishment of human colonic organoid knockout lines.

Human colonic organoids derived from biopsy or autopsy tissues are a vital tool to study mucosal homeostasis, model colonic diseases, and develop therapeutics. Rapid and reliable generation of knockout organoid lines from multiple donors enables analysis of specific gene functions. Here, we report protocols to produce colonic organoid knockout lines within 1 to 2 weeks using lentiviral delivery of CRISPR-Cas9, achieving knockout efficiency of 90% or greater. These lines are suitable for multi-lineage differentiation and downstream analysis. For complete details on the use and execution of this protocol, please refer to Gu et al. (2022).

Membrane Bound Peroxiredoxin-1 Serves as a Biomarker for In Vivo Detection of Sessile Serrated Adenomas.

Aim: Sessile serrated adenomas (SSAs) are premalignant lesions driven by the BRAFV600E mutation to give rise to colorectal cancers (CRCs). They are often missed during white light colonoscopy because of their subtle appearance. Previously, a fluorescently labeled 7mer peptide KCCFPAQ was shown to detect SSAs in vivo. We aim to identify the target of this peptide. Results: Peroxiredoxin-1 (Prdx1) was identified as the binding partner of the peptide ligand. In vitro binding assays and immunofluorescence staining of human colon specimens ex vivo supported this result. Prdx1 was overexpressed on the membrane of cells with the BRAFV600E mutation, and this effect was dependent on oxidative stress. RKO cells harboring the BRAFV600E mutation and human SSA specimens showed higher oxidative stress as well as elevated levels of Prdx1 on the cell membrane. Innovation and Conclusion: These results suggest that Prdx1 is overexpressed on the cell surface in the presence of oxidative stress and can serve as an imaging biomarker for in vivo detection of SSAs. Antioxid. Redox Signal. 36, 39-56.

UBCH5 Family Members Differentially Impact Stabilization of Mutant p53 via RNF128 Iso1 During Barrett's Progression to Esophageal Adenocarcinoma.

BACKGROUND & AIMS: TP53 mutations underlie Barrett's esophagus (BE) progression to dysplasia and cancer. During BE progression, the ubiquitin ligase (E3) RNF128/GRAIL switches expression from isoform 2 (Iso2) to Iso1, stabilizing mutant p53. However, the ubiquitin-conjugating enzyme (E2) that partners with Iso1 to stabilize mutant p53 is unknown. METHODS: Single-cell RNA sequencing of paired normal esophagus and BE tissues identified candidate E2s, further investigated in expression data from BE to esophageal adenocarcinoma (EAC) progression samples. Biochemical and cellular studies helped clarify the role of RNF128-E2 on mutant p53 stability. RESULTS: The UBE2D family member 2D3 (UBCH5C) is the most abundant E2 in normal esophagus. However, during BE to EAC progression, loss of UBE2D3 copy number and reduced expression of RNF128 Iso2 were noted, 2 known p53 degraders. In contrast, expression of UBE2D1 (UBCH5A) and RNF128 Iso1 in dysplastic BE and EAC forms an inactive E2-E3 complex, stabilizing mutant p53. To destabilize mutant p53, we targeted RNF128 Iso1 either by mutating asparagine (N48, 59, and 101) residues to block glycosylation to facilitate ß-TrCP1-mediated degradation or by mutating proline (P54 and 105) residues to restore p53 polyubiquitinating ability. In addition, either loss of UBCH5A catalytic activity, or disruption of the Iso1-UBCH5A interaction promoted Iso1 loss. Consequently, overexpression of either catalytically dead or Iso1-binding-deficient UBCH5A mutants destabilized Iso1 to degrade mutant p53, thus compromising the clonogenic survival of mutant p53-dependent BE cells. CONCLUSIONS: Loss of RNF128 Iso2-UBCH5C and persistence of the Iso1-UBCH5A complex favors mutant p53 stability to promote BE cell survival. Therefore, targeting of Iso1-UBCH5A may provide a novel therapeutic strategy to prevent BE progression.

Hepcidin sequesters iron to sustain nucleotide metabolism and mitochondrial function in colorectal cancer epithelial cells.

Colorectal cancer (CRC) requires massive iron stores, but the complete mechanisms by which CRC modulates local iron handling are poorly understood. Here, we demonstrate that hepcidin is activated ectopically in CRC. Mice deficient in hepcidin specifically in the colon tumour epithelium, compared with wild-type littermates, exhibit significantly diminished tumour number, burden and size in a sporadic model of CRC, whereas accumulation of intracellular iron by deletion of the iron exporter ferroportin exacerbates these tumour parameters. Metabolomic analysis of three-dimensional patient-derived CRC tumour enteroids indicates a prioritization of iron in CRC for the production of nucleotides, which is recapitulated in our hepcidin/ferroportin mouse CRC models. Mechanistically, our data suggest that iron chelation decreases mitochondrial function, thereby altering nucleotide synthesis, whereas exogenous supplementation of nucleosides or aspartate partially rescues tumour growth in patient-derived enteroids and CRC cell lines in the presence of an iron chelator. Collectively, these data suggest that ectopic hepcidin in the tumour epithelium establishes an axis to sequester iron in order to maintain the nucleotide pool and sustain proliferation in colorectal tumours.

Chromatin regulatory dynamics of early human small intestinal development using a directed differentiation model.

The establishment of the small intestinal (SI) lineage during human embryogenesis ensures functional integrity of the intestine after birth. The chromatin dynamics that drive SI lineage formation and regional patterning in humans are essentially unknown. To fill this knowledge void, we apply a cutting-edge genomic technology to a state-of-the-art human model of early SI development. Specifically, we leverage chromatin run-on sequencing (ChRO-seq) to define the landscape of active promoters, enhancers and gene bodies across distinct stages of directed differentiation of human pluripotent stem cells into SI spheroids with regional specification. Through comprehensive ChRO-seq analysis we identify candidate stage-specific chromatin activity states, novel markers and enhancer hotspots during the directed differentiation. Moreover, we propose a detailed transcriptional network associated with SI lineage formation or regional patterning. Our ChRO-seq analyses uncover a previously undescribed pattern of enhancer activity and transcription at HOX gene loci underlying SI regional patterning. We also validated this unique HOX dynamics by the analysis of single cell RNA-seq data from human fetal SI. Overall, the results lead to a new proposed working model for the regulatory underpinnings of human SI development, thereby adding a novel dimension to the literature that has relied almost exclusively on non-human models.

Ulcerative Colitis-Derived Colonoid Culture: A Multi-Mineral-Approach to Improve Barrier Protein Expression.

BACKGROUND: Recent studies demonstrated that Aquamin®, a calcium-, magnesium-rich, multi-mineral natural product, improves barrier structure and function in colonoids obtained from the tissue of healthy subjects. The goal of the present study was to determine if the colonic barrier could be improved in tissue from subjects with ulcerative colitis (UC). METHODS: Colonoid cultures were established with colon biopsies from 9 individuals with UC. The colonoids were then incubated for a 2-week period under control conditions (in culture medium with a final calcium concentration of 0.25 mM) or in the same medium supplemented with Aquamin® to provide 1.5 - 4.5 mM calcium. Effects on differentiation and barrier protein expression were determined using several approaches: phase-contrast and scanning electron microscopy, quantitative histology and immunohistology, mass spectrometry-based proteome assessment and transmission electron microscopy. RESULTS: Although there were no gross changes in colonoid appearance, there was an increase in lumen diameter and wall thickness on histology and greater expression of cytokeratin 20 (CK20) along with reduced expression of Ki67 by quantitative immunohistology observed with intervention. In parallel, upregulation of several differentiation-related proteins was seen in a proteomic screen with the intervention. Aquamin®-treated colonoids demonstrated a modest up-regulation of tight junctional proteins but stronger induction of adherens junction and desmosomal proteins. Increased desmosomes were seen at the ultrastructural level. Proteomic analysis demonstrated increased expression of several basement membrane proteins and hemidesmosomal components. Proteins expressed at the apical surface (mucins and trefoils) were also increased as were several additional proteins with anti-microbial activity or that modulate inflammation. Finally, several transporter proteins that affect electrolyte balance (and, thereby affect water resorption) were increased. At the same time, growth and cell cycle regulatory proteins (Ki67, nucleophosmin, and stathmin) were significantly down-regulated. Laminin interactions, matrix formation and extracellular matrix organization were the top three up-regulated pathways with the intervention. CONCLUSION: A majority of individuals including patients with UC do not reach the recommended daily intake for calcium and other minerals. To the extent that such deficiencies might contribute to the weakening of the colonic barrier, the findings employing UC tissue-derived colonoids here suggest that adequate mineral intake might improve the colonic barrier.

Low-Level Mouse DNA in Conditioned Medium Generates False Positive Cross-Species Contamination Results in Human Organoid Cultures.

Cell line authentication is critical for preventing the use of mixed or misidentified cell lines in research. Current efforts include short tandem repeat (STR) analysis and PCR-based assays to detect mixed species cultures. Using PCR analysis with mouse-specific primers, we identified contaminating mouse DNA in growth factor conditioned medium (CM) derived from the L-WRN cell line (L-WRN CM), as well as in human organoid cultures maintained in the L-WRN CM. DNA isolated from L-WRN CM matched the L-WRN cell signature by STR analysis. Organoid lines that were positive for murine DNA by PCR were further analyzed via bulk RNA-sequencing and transcripts were aligned to the human and mouse genomes. RNA analysis failed to detect mouse-specific gene expression above background levels, suggesting no viable murine cells were present in the organoid cultures. We interpret our data to show conclusive evidence that mouse cell-derived CM can be a source of contaminating murine DNA detected in human organoid cultures, even though live, transcriptionally-active murine cells are not present. Together, our findings suggest that multiple methods may be required to authenticate human organoid or cell lines and urges cautious interpretation of DNA-based PCR cell line authentication results.

Identification and Isolation of Human LGR5+ Cells Using an Antibody-Based Strategy.

Leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) has been identified as a marker of stem cells across multiple tissues. Lgr5-expressing cells are also regulators of tissue homeostasis and wound repair, and drivers of carcinogenic progression. The majority of information about Lgr5-expressing cells derives from genetically engineered mouse models. Human studies have been limited by a lack of specific reagents and experimental procedures for the purification of these cells. We recently demonstrated that antibody-based purification can be used to obtain viable LGR5-expressing cells from human primary tissues and patient derived organoids. Here, we provide detailed methods for the purification of these cells from colonic epithelial organoids generated from patient-derived tissues, from induced pluripotent stem cell (iPSC) derived intestinal organoids, and from freshly isolated patient tissue intestinal crypts. These methods will facilitate experimental analysis of human LGR5-expressing cells in development, wound healing, and cancer.

Differentiation of human colon tissue in culture: Effects of calcium on trans-epithelial electrical resistance and tissue cohesive properties.

BACKGROUND AND AIMS: Human colonoid cultures maintained under low-calcium (0.25 mM) conditions undergo differentiation spontaneously and, concomitantly, express a high level of tight junction proteins, but not desmosomal proteins. When calcium is included to a final concentration of 1.5-3.0 mM (provided either as a single agent or as a combination of calcium and additional minerals), there is little change in tight junction protein expression but a strong up-regulation of desmosomal proteins and an increase in desmosome formation. The aim of this study was to assess the functional consequences of calcium-mediated differences in barrier protein expression. METHODS: Human colonoid-derived epithelial cells were interrogated in transwell culture under low- or high-calcium conditions for monolayer integrity and ion permeability by measuring trans-epithelial electrical resistance (TEER) across the confluent monolayer. Colonoid cohesiveness was assessed in parallel. RESULTS: TEER values were high in the low-calcium environment but increased in response to calcium. In addition, colonoid cohesiveness increased substantially with calcium supplementation. In both assays, the response to multi-mineral intervention was greater than the response to calcium alone. Consistent with these findings, several components of tight junctions were expressed at 0.25 mM calcium but these did not increase substantially with supplementation. Cadherin-17 and desmoglein-2, in contrast, were weakly-expressed under low calcium conditions but increased with intervention. CONCLUSIONS: These findings indicate that low ambient calcium levels are sufficient to support the formation of a permeability barrier in the colonic epithelium. Higher calcium levels promote tissue cohesion and enhance barrier function. These findings may help explain how an adequate calcium intake contributes to colonic health by improving barrier function, even though there is little change in colonic histological features over a wide range of calcium intake levels.

Enteroendocrine Progenitor Cell-Enriched miR-7 Regulates Intestinal Epithelial Proliferation in an Xiap-Dependent Manner.

BACKGROUND & AIMS: The enteroendocrine cell (EEC) lineage is important for intestinal homeostasis. It was recently shown that EEC progenitors contribute to intestinal epithelial growth and renewal, but the underlying mechanisms remain poorly understood. MicroRNAs are under-explored along the entire EEC lineage trajectory, and comparatively little is known about their contributions to intestinal homeostasis. METHODS: We leverage unbiased sequencing and eight different mouse models and sorting methods to identify microRNAs enriched along the EEC lineage trajectory. We further characterize the functional role of EEC progenitor-enriched miRNA, miR-7, by in vivo dietary study as well as ex vivo enteroid in mice. RESULTS: First, we demonstrate that miR-7 is highly enriched across the entire EEC lineage trajectory and is the most enriched miRNA in EEC progenitors relative to Lgr5+ intestinal stem cells. Next, we show in vivo that in EEC progenitors miR-7 is dramatically suppressed under dietary conditions that favor crypt division and suppress EEC abundance. We then demonstrate by functional assays in mouse enteroids that miR-7 exerts robust control of growth, as determined by budding (proxy for crypt division), EdU and PH3 staining, and likely regulates EEC abundance also. Finally, we show by single-cell RNA sequencing analysis that miR-7 regulates Xiap in progenitor/stem cells and we demonstrate in enteroids that the effects of miR-7 on mouse enteroid growth depend in part on Xiap and Egfr signaling. CONCLUSIONS: This study demonstrates for the first time that EEC progenitor cell-enriched miR-7 is altered by dietary perturbations and that it regulates growth in enteroids via intact Xiap and Egfr signaling.

Astrovirus replication in human intestinal enteroids reveals multi-cellular tropism and an intricate host innate immune landscape.

Human astroviruses (HAstV) are understudied positive-strand RNA viruses that cause gastroenteritis mostly in children and the elderly. Three clades of astroviruses, classic, MLB-type and VA-type have been reported in humans. One limitation towards a better understanding of these viruses has been the lack of a physiologically relevant cell culture model that supports growth of all clades of HAstV. Herein, we demonstrate infection of HAstV strains belonging to all three clades in epithelium-only human intestinal enteroids (HIE) isolated from biopsy-derived intestinal crypts. A detailed investigation of infection of VA1, a member of the non-canonical HAstV-VA/HMO clade, showed robust replication in HIE derived from different patients and from different intestinal regions independent of the cellular differentiation status. Flow cytometry and immunofluorescence analysis revealed that VA1 infects several cell types, including intestinal progenitor cells and mature enterocytes, in HIE cultures. RNA profiling of VA1-infected HIE uncovered that the host response to infection is dominated by interferon (IFN)-mediated innate immune responses. A comparison of the antiviral host response in non-transformed HIE and transformed human colon carcinoma Caco-2 cells highlighted significant differences between these cells, including an increased magnitude of the response in HIE. Additional studies confirmed the sensitivity of VA1 to exogenous IFNs, and indicated that the endogenous IFN response of HIE to curtail the growth of strains from all three clades. Genotypic variation in the permissiveness of different HIE lines to HAstV could be overcome by pharmacologic inhibition of JAK/STAT signaling. Collectively, our data identify HIE as a universal infection model for HAstV and an improved model of the intestinal epithelium to investigate enteric virus-host interactions.

Dietary polyunsaturated fatty acids modulate adipose secretome and is associated with changes in mammary epithelial stem cell self-renewal.

Chronic low-grade adipose inflammation, characterized by aberrant adipokine production and pro-inflammatory macrophage activation/polarization is associated with increased risk of breast cancer. Adipocyte fatty acid composition is influenced by dietary availability and may regulate adipokine secretion and adipose inflammation. After feeding F344 rats for 20 weeks with a Western diet or a fish oil-supplemented diet, we cultured primary rat adipose tissue in a three-dimensional explant culture and collected the conditioned medium. The rat adipose tissue secretome was assayed using the Proteome Profiler Cytokine XL Array, and adipose tissue macrophage polarization (M1/M2 ratio) was assessed using the iNOS/ARG1 ratio. We then assessed the adipokine's effects upon stem cell self-renewal using primary human mammospheres from normal breast mammoplasty tissue. Adipose from rats fed the fish oil diet had an ω-3:ω-6 fatty acid ratio of 0.28 compared to 0.04 in Western diet rats. The adipokine profile from the fish oil-fed rats was shifted toward adipokines associated with reduced inflammation compared to the rats fed the Western diet. The M1/M2 macrophage ratio decreased by 50% in adipose of fish oil-fed rats compared to that from rats fed the Western diet. Conditioned media from rats fed the high ω-6 Western diet increased stem cell self-renewal by 62%±9% (X¯%±SD) above baseline compared to only an 11%±11% increase with the fish oil rat adipose. Modulating the adipokine secretome with dietary interventions therefore may alter stromal-epithelial signaling that plays a role in controlling mammary stem cell self-renewal.

Calcium-induced differentiation in normal human colonoid cultures: Cell-cell / cell-matrix adhesion, barrier formation and tissue integrity.

BACKGROUND AND AIMS: The goal of the study was to assess calcium alone and Aquamin, a multi-mineral natural product that contains magnesium and detectable levels of 72 trace elements in addition to calcium, for capacity to affect growth and differentiation in colonoid cultures derived from histologically-normal human colon tissue. METHODS: Colonoid cultures were maintained in a low-calcium (0.25 mM) medium or in medium supplemented with an amount of calcium (1.5-3.0 mM), either from calcium alone or Aquamin for a period of two weeks. This was shown in a previous study to induce differentiation in colonoids derived from large adenomas. Changes in growth, morphological features and protein expression profile were assessed at the end of the incubation period using a combination of phase-contrast and scanning electron microscopy, histology and immunohistology, proteomic assessment and transmission electron microscopy. RESULTS: Unlike the previously-studied tumor-derived colonoids (which remained un-differentiated in the absence of calcium-supplementation), normal tissue colonoids underwent differentiation as indicated by gross and microscopic appearance, a low proliferative index and high-level expression of cytokeratin 20 in the absence of intervention (i.e., in control condition). Only modest additional changes were seen in these parameters with either calcium alone or Aquamin (providing up to 3.0 mM calcium). In spite of this, proteomic analysis and immunohistochemistry revealed that both interventions induced strong up-regulation of proteins that promote cell-cell and cell-matrix adhesive functions, barrier formation and tissue integrity. Transmission electron microscopy revealed an increase in desmosomes in response to intervention. CONCLUSIONS: These findings demonstrate that colonoids derived from histologically normal human tissue can undergo differentiation in the presence of a low ambient calcium concentration. However, higher calcium levels induce elaboration of proteins that promote cell-cell and cell-matrix adhesion. These changes could lead to improved barrier function and improved colon tissue health.